Impact of the isoelectric point of model parvoviruses on viral retention in anion-exchange chromatography.

Anion-exchange chromatography (AEX) is used in the downstream purification of monoclonal antibodies to remove impurities and potential viral contamination based on electrostatic interactions. Although the isoelectric point (pI) of viruses is considered a key factor predicting the virus adsorption to the resin, the precise molecular mechanisms involved remain unclear. To address this question, we compared structurally homologous parvoviruses that only differ in their surface charge distribution. A single charged amino acid substitution on the capsid surface of minute virus of mice (MVM) provoked an increased apparent pI (pIapp ) 6.2 compared to wild-type MVM (pIapp = 4.5), as determined by chromatofocusing. Despite their radically different pIapp , both viruses displayed the same interaction profile in Mono Q AEX at different pH conditions. In contrast, the closely related canine parvovirus (pIapp = 5.3) displayed a significantly different interaction at pH 5. The detailed structural analysis of the intricate three-dimensional structure of the capsids suggests that the charge distribution is critical, and more relevant than the pI, in controlling the interaction of a virus with the chromatographic resin. This study contributes to a better understanding of the molecular mechanisms governing virus clearance by AEX, which is crucial to enable robust process design and maximize safety.

Characterizing and enhancing virus removal by protein A chromatography.

Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal.

The effects of buffer condition on the fouling behavior of MVM virus filtration of an Fc-fusion protein.

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc-fusion protein using the three commercially available filters, F-1, F-2, and F-3 in a range of buffer conditions including sodium-phosphate and tris-acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions.

A field strain of minute virus of mice (MVMm) exhibits age- and strain-specific pathogenesis.

The influence of mouse strain, immune competence and age on the pathogenesis of a field strain of minute virus of mice (MVMm) was examined in BALB/c, C3H, C57BL/6 and SCID mice experimentally infected as neonates, weanlings and adults. Sera, bodily excretions and tissues were harvested at 7, 14, 28 and 56 days after inoculation and evaluated by serology, quantitative PCR and histopathology. Seroconversion to recombinant viral capsid protein 2 was consistently observed in all immunocompetent strains of mice, regardless of the age at which they were inoculated, while seroconversion to the viral nonstructural protein 1 was only consistently detected in neonate inoculates. Viral DNA was detected by quantitative PCR in multiple tissues of immunocompetent mice at each time point after inoculation, with the highest levels being observed in neonate inoculates at 7 days after inoculation. In contrast, viral DNA levels in tissues and bodily excretions increased consistently over time in immunodeficient SCID mice, regardless of the age at which they were inoculated, with mortality being observed in neonatal inoculates between 28 and 56 days after inoculation. Overall, productive infection was observed more frequently in immunocompetent mice inoculated as neonates as compared to those inoculated as weanlings or adults, and immunodeficient SCID mice developed persistent, progressive infection, with mortality being observed in mice inoculated as neonates. Importantly, the clinical syndrome observed in experimentally infected SCID neonatal mice recapitulates the clinical presentation reported for the naturally infected immunodeficient NOD µ-chain knockout mice from which MVMm was initially isolated.

Anion exchange membrane adsorbers for flow-through polishing steps: Part I. Clearance of minute virus of mice.

Membrane adsorbers may be a viable alternative to the packed-bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18-22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands.

Anion exchange membrane adsorbers for flow-through polishing steps: Part II. Virus, host cell protein, DNA clearance, and antibody recovery.

Anion exchange membrane adsorbers are used for contaminant removal in flow-through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion-exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0-200 mM NaCl, pH 6.0-9.0, and flow rates of 4-20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion-exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow-through polishing steps.

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: part II. DNA and virus separations.

The surface-initiated polymerization protocol developed in part I was used to prepare strong anion-exchange membranes with variable polymer chain graft densities and degrees of polymerization for DNA and virus particle separations. A focus of part II was to evaluate the role of polymer nanolayer architecture on DNA and virus binding. Salmon sperm-DNA (SS-DNA) was used as model nucleic acid to measure the dynamic-binding capacities at 10% breakthrough. The dynamic-binding capacity increases linearly with increasing poly ([2-(methacryloyloxy)ethyl]trimethylammonium chloride) chain density up to the highest chain density used in this study. The new membranes yielded threefold higher SS-DNA-binding capacity (30 mg/mL) than a leading commercial membrane with the same functional group chemistry. Elution of bound DNA yielded a sharp peak, and resulted in a 13-fold increase relative to the feed concentration. This concentration effect further demonstrates the highly favorable transport properties of the newly designed Q-type membranes. However, unlike findings in part I on protein binding, SS-DNA binding was not fully reversible. Minute virus of mice (MVM) was used as model virus to evaluate the virus clearance performance of newly designed Q-type membranes. Log reduction of virus (LRV) of MVM increased with increasing polymer chain density. Membranes exhibited >4.5 LRV for the given MVM impurity load and may be capable of higher LRV values, as the MVM concentration in the flow-through fraction of these samples was below the limit of detection of the assay.

Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development.

Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates.

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.

Lack of transmission of mouse minute virus (MMV) from in vitro-produced embryos to recipients and pups due to the presence of cumulus cells during the in vitro fertilization process.

The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (10(4) TCID(50) [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 10(5) cumulus cells were analyzed 5 h after exposure to 10(4) TCID(50)/ml MMVp, cells had an average titer of 10(4) TCID(50)/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.

PEG enhances viral clearance on ceramic hydroxyapatite.

Viral clearance across ceramic hydroxyapatite (CHT) was examined in two elution systems: sodium chloride and sodium chloride plus poly(ethylene glycol) (PEG). In both cases clearance of xenotropic murine leukemia virus was significant (3-4 log) while that of minute virus of mice varied between 1.7 and 2.7 log; in addition, the addition of PEG to the elution buffer enhanced viral clearance. The data are in agreement with the previous results and demonstrate that additional clearance can be obtained by adding PEG to a ceramic hydroxyapatite buffer system.

Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production.

Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log(10) clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6log(10) dynamic range with a limit of detection (LOD) of approximately 1 genome copy/microL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.

Evaluation of viral removal by nanofiltration using real-time quantitative polymerase chain reaction.

Nanofiltration is commonly introduced into purification processes of biologics produced in mammalian cells to serve as a designated step for removal of potential exogenous viral contaminants and endogenous retrovirus-like particles. The LRV (log reduction value) achieved by nanofiltration is often determined by cell-based infectivity assay, which is time-consuming and labour-intensive. We have explored the possibility of employing QPCR (quantitative PCR) to evaluate LRV achieved by nanofiltration in scaled-down studies using two model viruses, namely xenotropic murine leukemia virus and murine minute virus. We report here the successful development of a QPCR-based method suitable for quantification of virus removal by nanofiltration. The method includes a nuclease treatment step to remove free viral nucleic acids, while viral genome associated with intact virus particles is shielded from the nuclease. In addition, HIV Armored RNA was included as an internal control to ensure the accuracy and reliability of the method. The QPCRbased method described here provides several advantages such as better sensitivity, faster turnaround time, reduced cost and higher throughput over the traditional cell-based infectivity assays.

Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications.

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Differentiation of minute virus of mice and mouse parvovirus by high resolution melting curve analysis.

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/µL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV.

Amino Acid Side Chains Buried along Intersubunit Interfaces in a Viral Capsid Preserve Low Mechanical Stiffness Associated with Virus Infectivity.

Single-molecule experimental techniques and theoretical approaches reveal that important aspects of virus biology can be understood in biomechanical terms at the nanoscale. A detailed knowledge of the relationship in virus capsids between small structural changes caused by single-point mutations and changes in mechanical properties may provide further physics-based insights into virus function; it may also facilitate the engineering of viral nanoparticles with improved mechanical behavior. Here, we used the minute virus of mice to undertake a systematic experimental study on the contribution to capsid stiffness of amino acid side chains at interprotein interfaces and the specific noncovalent interactions they establish. Selected side chains were individually truncated by introducing point mutations to alanine, and the effects on local and global capsid stiffness were determined using atomic force microscopy. The results revealed that, in the natural virus capsid, multiple, mostly hydrophobic, side chains buried along the interfaces between subunits preserve a comparatively low stiffness of most (S2 and S3) regions. Virtually no point mutation tested substantially reduced stiffness, whereas most mutations increased stiffness of the S2/S3 regions. This stiffening was invariably associated with reduced virus yields during cell infection. The experimental evidence suggests that a comparatively low stiffness at S3/S2 capsid regions may have been biologically selected because it facilitates capsid assembly, increasing infectious virus yields. This study demonstrated also that knowledge of individual amino acid side chains and biological pressures that determine the physical behavior of a protein nanoparticle may be used for engineering its mechanical properties.

Antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (PCR) of faecal samples.

Parvoviruses remain one of the most common viral infections seen in laboratory mouse colonies. The purpose of this study was to develop an antemortem polymerase chain reaction (PCR) assay to detect mice infected with mouse parvovirus-1 (MPV) and mice minute virus (MMV) using faecal samples. The MMV PCR assay consistently detected as few as 100 plasmid copies of MMV in faecal samples, while the MPV PCR assay detected as few as 10 plasmid copies of MPV. Faecal pellets from infected mice held at room temperature from 1 to 7 days tested positive by MMV and MPV PCR, respectively. This demonstrates that parvovirus DNA is stable in faecal samples kept at room temperature. PCR assays were also used to follow the length of MMV and MPV shedding in faeces from SENCAR mice, which were endemically infected with multiple agents. MMV faecal shedding was detected in 60-70% of the mice 5-7 weeks old, and by 13 weeks of age, faecal samples from all mice were negative for MMV. MPV faecal shedding was detected in 90-100% of the mice 5-11 weeks old; however, by 19 weeks of age, faecal samples from all mice were negative for MPV. These findings confirm that faecal shedding occurs for a limited time and suggest that 5-9-week-old mice are the most appropriate age group in endemically infected mice for faecal testing by MMV and MPV PCR.

Detection of rat parvovirus type 1 and rat minute virus type 1 by polymerase chain reaction.

Two newly recognized parvovirus species, rat parvovirus 1 (RPV-1) and rat minute virus 1 (RMV-1), were recently identified in naturally infected rats. In this study, two polymerase chain reaction (PCR) assays were developed to specifically detect RPV-1 and RMV-1. The RPV-1 PCR assay amplified the expected 487-bp deoxyribonucleic acid (DNA) fragment only in the presence of RPV-1 DNA; the RMV-1 PCR assay amplified the expected 843-bp product only from RMV-1 DNA, not from other rodent parvoviruses. The RPV-1 and the RMV-1 PCR assays detected approximately 18 and 70 copies of DNA template, respectively. These two PCR assays were shown to be sensitive, specific and rapid methods for detecting RPV-1 and RMV-1 infections in rats. These assays may also be valuable for evaluation of biological specimens for parvovirus contamination.

Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing.

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.

In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice.

OBJECTIVE: Intranasal inoculation of the i strain of the parvovirus minute virus of mice (MVMi) into immunodeficient SCID mice induces suppression of myeloid and erythroid progenitors in the bone marrow (BM) and lethal leukopenia. In the present study, we investigated whether the mouse megakaryocytic lineage was susceptible to MVMi. MATERIALS AND METHODS: In vitro and in vivo infections with purified MVMi were conducted and their effects on the megakaryocytic lineage studied. RESULTS: In vitro infection of BM cells showed a multiplicity of infection-dependent inhibition in the colony-forming ability of megakaryocytic progenitors (colony-forming unit megakaryocyte [CFU-MK]). Neutralization or heat inactivation of the virus abrogated this inhibition. Expression of the MVMi nonstructural-1 protein was detected in the in vitro infected and cultured megakaryocytic cells. In vivo, intranasal inoculation of a lethal dose of virus was incapable of producing significant thrombocytopenia, although an increase in mean platelet volume was observed. Significantly, in the BM of these animals, a progressive decrease in CFU-MK was noted from day 14 postinfection, with survival rates less than 1% by day 35 postinfection. At day 35 postinfection, intermediate megakaryocytic differentiation stages showed maintenance of the proportion and ploidy of cells and a moderate decrease in the total number of these cells per femoral BM. CONCLUSIONS: The results demonstrate that MVMi is capable of inhibiting the proliferative capacity of megakaryocytic committed progenitors both in vitro and in vivo. Moreover, the in vivo data show that depletion of BM CFU-MK is compensated by the system, and platelet counts in the peripheral blood are maintained close to normal values.